The present invention relates to two new microorganism strains of Saccharothrix, designated as YS-44442 and YS-45494, a process of producing pravastatin using the strains, and an improved process for isolation of (HMG)-CoA reductase inhibitors.
It has been recognized that an elevated blood cholesterol level is one of the major risk factors to atherosclerotic diseases, specifically to coronary heart diseases. The monitor for the cholesterol biosynthesis is very helpful to control the diseases. 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase is the rate-limiting enzyme in the cholesterol biosynthesis. By inhibiting the activity of (HMG)-CoA reductase, blood cholesterol levels in the bodies can be effectively reduced.
A number of (HMG)-CoA reductase inhibitors have been discovered, such as pravastatin, compactin, lovastatin. They have the following formula in the lactone form and may exist in other forms such as the acid form or and the salts and esters thereof. 
These (HMG)-CoA reductase inhibitors are very effective in lowering blood cholesterol level in most animals including human. Pravastatin is even more active than compactin and lovastatin, and has been applied in the treatment of hypercholesterolemia (Nara, F et al. Curr. Genet. 23: 28-32 (1993)).
Pravastatin is a 3 xcex2-hydroxy derivative of compactin. It has been reported that pravastatin is produced by converting compactin through microbial hydroxylation using various genera of fungi and bacteria, such as Streptomyces roseochrornogenus (U.S. Pat. No. 4,346,277) and Actinomadura sp. (Peng, M. et al., J. Antibiotics, December: 1032-1035 (1997), and Peng Y. and A. L. Demain, J. Mol. Cataly. B: Enzymatic 10: 151-156 (2000)). However, these fungi or bacteria do not tolerate a high amount of compactin added in the fungal or bacterial fermentation broth, likely due to the anti-fungal activity of compactin, and thus exhibit low productivity of pravastatin. Therefore, there is a need to find a new microorganism which is tolerable to a higher amount of compactin and has effective conversion activity.
In general, the isolation of (HMG)-CoA reductase inhibitors from a fermentation broth is conducted by serious procedures of extraction, chromatography, lactonization and crystallization process. EP 0 877 089A1 discloses a (HMG)-CoA reductase inhibitor preparation process, wherein a fermentation broth containing the inhibitor (e.g., lovastatin) is basified prior to filtration to remove the cells and then the filtrate was loaded through a column. The eluate was extracted with toluene and subsequently the lactonization is conducted to produce the inhibitor.
When using chromatography, however, a large column and a great volume of fermentation broth containing (HMG)-CoA reductase inhibitors are usually needed to obtain a desired yield of the inhibitors, thereby increasing the difficulty in handling the purification process of the inhibitors. Furthermore, a lactonization reaction usually needs much energy and time. Therefore, there is a need to seek an improved process of obtaining a (HMG)-CoA reductase inhibitor in a good yield and purity without proceeding a lactonization reaction and using a chromatography, and so as to reduce the cost.
One object of the invention is to provide two new microorganism strains of Saccharothrix capable of producing pravastatin designated as YS-44442 and YS-45494 as well as their mutants.
Another object of the invention is to provide a process of producing pravastatin by using the microorganisms of the invention. In particular, the process of producing pravastatin comprises the steps of (a) cultivating YS-44442 or YS-45494 at a suitable condition to generate a fermentation broth; (b) feeding compactin into the broth; (c) fermenting the broth for a period of time to convert the compactin to pravastatin; (d) isolating the pravastatin from the broth.
Still another object of the invention is to provide a process of isolating a (HMG)-CoA reductase inhibitor comprising the steps of (1) adding an ammonium sulfate into a first solution containing the (HMG)-CoA reductase inhibitor to produce a precipitation; (2) isolating the precipitation; (3) dissolving the precipitation with a polar solvent to produce a second solution; (4) adjusting the pH of the second solution to about 4 to about 6; and (5) extracting the second solution with an water immiscible solvent to isolate the (HMG)-CoA reductase inhibitor. The preparation process further comprises a step of reacting the isolated (HMG)-CoA reductase inhibitor with an organic or inorganic cation source to generate a salt form of the inhibitor.